The Process of Protein Synthesis
Protein synthesis is one of the most central life processes. A protein is formed by the linkage of multiple amino acids via peptide bonds, according to a sequence defined by the template messenger RNA (mRNA). Protein synthesis occurs in the ribosomes, the protein manufacturing plants of every organism and nearly every cell type.
Ribosomes are ribonucleoprotein particles consisting of a small and large subunit. In bacteria these subunits have sedimentation coefficients of 30 and 50, and thus are referred to as “30S” and “50S” respectively; in eukaryotes the sedimentation coefficients are 40 and 60. The translation system makes use of a large number of components, including inter alia the ribosome, initiation, elongation, termination and recycling factors, transfer RNA, amino acids, aminoacyl synthetases, magnesium, and the product polypeptides.
tRNAs are 73-93 nucleoside RNA molecules that recruit amino acid residues to the protein synthesis machinery. The structure of tRNA is often depicted as a cloverleaf representation. Structural elements of a typical tRNA include an acceptor stem, a D-loop, an anticodon loop, a variable loop and a TψC loop. Aminoacylation, or charging, of tRNA results in linking the carboxyl terminal of an amino acid to the 2′-(or 3′-) hydroxyl group of a terminal adenosine base via an ester linkage. Aminoacylation occurs in two steps, amino acid activation (i.e. adenylation of the amino acid to produce aminoacyl-AMP), tRNA aminoacylation (i.e. attachment of an amino acid to the tRNA).
Protein translation, also referred to as “polypeptide synthesis,” begins by formation of the initiation complex, composed of the two ribosomal subunits, proteins known as “initiation factors,” mRNA, and an initiator tRNA, which recognizes the base sequence UAG, i.e. the initiator codon of open reading frames. Initiation factors are proteins whose function is to bring the mRNA and initiator tRNA to the ribosome. The initiation factors first bind to the small ribosome subunit, then to the initiator tRNA, and then the large ribosomal subunit is recruited. Elongation proceeds with repeated cycles of charged tRNAs binding to the ribosome (a step termed “recognition”), peptide bond formation, and translocation. Elongation factors recruit and assist with binding of additional tRNAs and in elongation of the polypeptide chain. Elongation utilizes enzymes such as peptidyl transferase, which catalyzes addition of amino acid moieties onto the growing chain. Termination factors recognize a stop signal, such as the base sequence UGA, in the mRNA, terminating polypeptide synthesis and releasing the polypeptide chain and mRNA from the ribosome (Kapp et al., 2004, Annu Rev Biochem. 73:657-704). After termination of translation, the recycling factor enables the ribosome to dissociate into its two separate subunits, which are then available for a new round of protein synthesis.
In eukaryotes, ribosomes are often attached to the membranes of the endoplasmic reticulum (ER) and Golgi compartments. Additionally, ribosomes are active in organelles such as endoplasmic reticulum and mitochondria and, in plant cells, in chloroplasts, and other subcellular compartments. One important locus of protein synthesis activity is the dendritic spines of neurons.
Ribosomes as Targets of Drugs and Antibiotic Compounds
There are variations between eukaryotic and prokaryotic translation mechanisms, as well as subtler differences between eukaryotic ribosomes in different organisms and subcellular components. Prokaryotic ribosomes are the targets of many antibiotic compounds (Yonath, Annu Rev Biochem. 74:649-79, 2005; Hainrichson M et al, Designer aminoglycosides: the race to develop improved antibiotics and compounds for the treatment of human genetic diseases. Org Biomol Chem 6 (2):227-39, 2008). Such antibiotics must not exhibit significant inhibition of eukaryotic ribosomes, including mitochondrial ribosomes, and thus may exploit subtle differences between prokaryotic vs. mammalian and mitochondrial ribosomes. Widespread use of antibiotics over the past half-century has lead to emergence of bacterial strains resistant to many antibiotics now in use.
For these reasons, fast and accurate measurement of ribosomal activity is important for development of new types of antibiotics, including activity of mitochondrial ribosomes in the context of an intact eukaryotic cell, in order to produce new antibiotics to combat the increasing number of the antibiotic-resistant strains (Cohen, 1992, Science, 257: 1050-1055). Use of these assays may lead to the discovery of new classes of antibiotics that are toxic to a broad range of pathogenic bacteria, and at the same time, harmless to their mammalian hosts.
Diseases Related to Protein Translation
Control of protein translation is implicated in a large number of diseases. For example, a family of central nervous system (CNS) disorders connected with protein synthesis disturbances in neural spines is currently the subject of intense research. The family includes fragile X mental retardation, autism, aging and memory degeneration disorders such as Alzheimer's disease. Neural spines and synapses contain their own protein synthesis machinery. Synaptic plasticity, underpinning the most basic neural functions of memory and learning, is dependent upon proper regulation of spinal protein synthesis. Memory and aging are hypothesized to be linked to this phenomenon; fragile-X mental retardation and autism are known to be.
Fragile-X syndrome is the most common form of inherited mental retardation in humans. Conditions associated with the syndrome include mild to moderate cognitive abnormalities and behavioral disorders similar to autism, attention deficit disorder, obsessive-compulsive tendencies, hyperactivity, slow development of motor skills, and anxiety/fear disorder. Fragile X syndrome results from a deficiency of the fragile X mental retardation protein, FMRP, which is encoded by the X-linked FMR1 gene, usually due to transcriptional silencing of this gene brought about by the expansion and hypermethylation of a (CGG)n trinucleotide repeat in the 5′ untranslated region (UTR) of the gene, indicating that the necessity of FMRP for higher cognitive function. In the cytoplasm, FMRP-mRNP is normally associated with translating polyribosomes. In dendrites, FMRP is believed to modulate translation of mRNAs and acts as a translational suppressor.
Another important family of diseases directly connected to protein synthesis includes genetic disorders associated with the presence of premature termination codons (PTC) in the coding sequence of a critical protein, preventing its translation. Such diseases include Duchenne Muscular Dystrophy and a large family of congenital diseases. A small molecule known as PTC124 (Welch E M et al, Nature 2007 May 3; 447(7140):87-91) helps the ribosome slide over the mutated codon, thereby producing the required protein, albeit at only at 1-5% of normal concentrations. These amounts are often sufficient to sustain the life of an afflicted individual. PTC suppression has also been achieved by introducing charged suppressor tRNA into a living cell, enabling readthrough suppression of the PTC-containing mRNA and accumulation of the encoded protein (Sako et al, Nucleic Acids Symp Ser, 50:239-240, 2006.
Other diseases believed to be connected to control of protein synthesis include cardiac hypertrophy, restenosis, diabetes and obesity. Inflammatory bowel disease (e.g., ulcerative colitis and Crohn's disease) is associated with increased whole-body protein turnover. Reduced translational activity in cells, tissues, organs and organisms is a widely observed age-associated biochemical change. The consequences of slower rates of protein synthesis are manifold in the context of aging and age-related pathology. These include decreased availability of enzymes, inefficient removal of intracellular damaged substances, inefficient intra- and intercellular communication, decreased production of hormones and growth factors, decreased production of antibodies, and altered nature of the extracellular matrix.
In addition, control of protein synthesis is often compromised by cellular transformation. Novel anticancer drugs capable of targeting the ribosome in cancer cells are currently being developed (Palakurthi, S. S. et al., Cancer Research 61: 6213-6218, 2001).
Mitochondria-Related Diseases
Mitochondria found in eukaryotic cells have transcription and translation systems for expression of the endogenous mitochondrial DNA (mtDNA) that use a genetic code different from the universal code used by nuclear genomic DNA. Most mitochondrial proteins are encoded by nuclear DNA that is transcribed, translated in the cytosol, and imported into the mitochondria. However, some mitochondrial proteins are transcribed from mtDNA and translated within the organelle itself, using the mitochondrial system that includes two ribosomal RNA and 22 tRNAs. The human mitochondrial DNA (mtDNA) consists of 37 genes (Wallace, Gene. 354:169-80, 2005). The mitochondrial DNA encodes proteins that are essential components of the mitochondrial energy generation pathway, oxidative phosphorylation (OXPHOS). Oxidative phosphorylation generates heat to maintain body temperature and ATP to power cellular metabolism. Mitochondria also produce a significant fraction of cellular reactive oxygen species (ROS) and can initiate apoptosis through activation of the mitochondrial permeability transition pore (mtPTP) in response to energy deficiency and oxidative damage. Mitochondrial ROS cause mutation of mtDNA, which has been associated with a wide range of age-related diseases including neurodegenerative diseases, cardiomyopathy, metabolic diseases such as diabetes, and various cancers.
FRET, Quenching Pairs, and FCS
Fluorescence resonance energy transfer (FRET) is a method widely used to monitor biological interactions. FRET utilizes a donor fluorophore, having an emission spectrum that overlaps with the excitation spectrum of the acceptor fluorophore. Only when the donor fluorophore and acceptor fluorophore are in close proximity, typically about 10 nm, is a signal emitted from the acceptor fluorophore. FRET is described in Szöllosi J, Damjanovich S, Mátyus L, Application of fluorescence resonance energy transfer in the clinical laboratory: routine and research, Cytometry 34 (4):159-79, 1998. A quenching pair is a fluorophore in combination with a second molecule that quenches fluorescence of the fluorophore when in close proximity thereto. Thus, when the quenching pair is separated, under conditions wherein the fluorophore emits radiation, a signal is emitted.
Fluorescent Correlation Spectroscopy (FCS) is described for example in Schwille et al., Biophysical Journal, Vol. 77, 1999: 2251-2265; Wiseman and Petersen, Biophysical Journal, Vol. 76, 1999: 963-977; and Thompson et al., Current Opinion in Structural Biology, 2002, 12:634-641. In this method, signal variation is measured and used for computing basic parameters of the system, such as the number of fluorescing molecules in the system. The variability is mainly a function of molecules entering and leaving the illuminated volume.
Existing Methods of Measuring Protein Translation
Methods current used in the art typically comprise radioactive labeling of amino acid residues, following by electrophoretic separation of the protein mixture and detection of radioactive label. Such methods produce an estimation of the total production of proteins over a given period of time measured in minute, hours or days, as opposed to the instant readout of methods of the present invention. Current methods do not provide real-time measurements of the ribosomal activity, nor can they identify subcellular localization of protein synthesis or measure the dynamics of this activity.
U.S. Pat. No. 6,210,941 discloses methods for the non-radioactive labeling, detection, quantitation and isolation of nascent proteins translated in a cellular or cell-free translation system. tRNA molecules are mis-aminoacylated with non-radioactive markers that may be non-native amino acids, amino acid analogs or derivatives, or substances recognized by the protein synthesizing machinery. These methods require elaborate and expensive cell preparations and equipment to enable isolation of nascent proteins, and are not suitable as a simple tool for measuring general protein synthesis rates in live cells or organelles, particularly in real time.
U.S. Patent application Nos. 2003/0219783 and 2004/0023256 of Puglisi disclose compositions and methods for solid surface translation, where translationally competent ribosome complexes are immobilized on a solid surface. The ribosomes may be labeled to permit analysis of single molecules for determination of ribosomal conformational changes and translation kinetics. One or more components of the ribosome complex may be labeled at specific positions, and arrays of ribosome complexes may comprise a panel of different labels and positions of labels. Monitoring may comprise co-localization of fluorescently-labeled tRNA with fluorescently-labeled ribosomes or fluorescence resonance energy transfer (FRET) between a labeled ribosome and separately labeled mRNA. However, only cell-free translation methods are disclosed; methods for measuring overall cellular translation activity, in real time in viable cells or organelles, are neither disclosed nor suggested.
WO2004/050825 of the inventor of the present invention discloses methods for monitoring the synthesis of proteins by ribosomes in cells or a cell-free translation system. WO2005/116252 of the inventor of the present invention discloses methods for identifying proteins synthesized in a cell-free translation system. According to the methods described in these applications, the ribosome is engineered to carry a donor fluorophore, and tRNA, amino acids, and/or another component of the ribosome act as a fluorophore acceptor, via either their natural fluorescent properties or introduction of an engineered acceptor fluorophore. Illumination of ribosomes by a light source during translation excites the donor fluorophores and thereby the acceptor fluorophores whenever these are in sufficient proximity to a donor. One or a small number of ribosomes are typically analyzed in one batch. Neither of these references discloses or suggests the methods of the present invention for measuring overall cellular translation activity, in real time in viable cells or organelles.
There is an ongoing need for methods that provide a measure of overall cellular translation activity, in real time and in viable cells. Methods for measuring changes in protein synthesis rates in response to a drug candidate will be very useful for drug screening and assays for predicting therapeutic activity of candidate drugs. Also highly advantageous would be real-time measurement of ribosomal activity at sub-cellular resolution. The present invention overcomes problems and disadvantages associated with current strategies and provides methods for labeling, detection, and quantitation of general translation activity in real time.